rabbit anti-mt-nd2 polyclonal antibody pa5-103952  (Thermo Fisher)

Journal: Acta Neuropathologica

Article Title: Mitochondrial bioenergetic deficits in C9orf72 amyotrophic lateral sclerosis motor neurons cause dysfunctional axonal homeostasis

doi: 10.1007/s00401-020-02252-5

Figure Lengend Snippet: Transcriptomic analysis revealed reduced gene expression of the mitochondrial electron transport chain in human iPSC-derived C9orf72 -MNs. a Results of differential gene expression analysis, using DESeq2, examining the intersection in commonly and concordantly differentially expressed genes between the two mutant-isogene pairs, using an FDR of 20%, and discarding genes with an average FPKM < 1. The scatter plot shows the comparison of gene expression (as average FPKM). Red and blue data points denote the overlap of significantly up- and down-regulated genes in both mutant-correction pairs, respectively. b Summary of notable results from gene ontology analysis performed on the differentially expressed genes that identified putatively up (red) and down (blue) regulated altered pathways or processes. Average FPKM scatter plots comparing gene expression in C9orf72 mutant versus isogenic controls for two gene sets of interest ( c , mitochondrially encoded mitochondrial transcripts and ( d ) nuclear-encoded mitochondrial transcripts). Competitive gene set testing using CAMERA, examining the differential gene expression for these two gene sets of interest compared with all other genes, showed that mitochondrially encoded transcripts were down-regulated ( p = 7.39 × 10 –20 ), whereas nuclear-encoded mitochondrial transcripts were not ( p = 0.51). e Heatmap summarising the average gene expression (FPKM) of significantly dysregulated genes of complex I (MT-ND1, MT-ND2, MT-ND4, MT-ND4L, MT-ND5) and complex IV (MT-CO2, MT-CO3) subunits of the electron transport chain for C9orf72 versus isogenic control MNs. f Log 10 mean fold change ± SEM of gene expression normalised to the respective isogenic control determined via RT-PCR for C9-1 pair (black bars), C9-2 pair (red bars), and C9-3 pair (green bars) for MT-ND2 and MT-ND4 subunits of complex I; MT-CO2 and MT-CO3 subunits of complex IV; and MT-ATP6 subunit of complex V. g Representative image of western blot of protein lysates from two independent controls [Con-1, Con-2], three independent patient-derived C9orf72 lines [C9-1, C9-2, C9-3] with corresponding isogenic controls [C9-1Δ, C9-2Δ, C9-3Δ] using a primary Total OXPHOS cocktail antibody against multiple mitochondrial electron transport chain subunits. Bands depict protein expression of complex I (NDUFB8; reduced), complex II (SDHB), complex III (UQCRC2), complex IV (MT-CO2; reduced) and complex V (ATP5A), and housekeeper proteins against GAPDH and mitochondrial outer membrane protein, VDAC-1

Article Snippet: Sections were dried overnight at 40 °C and immunostaining was performed, following epitope retrieval in citric acid buffer (pH 6) in a pressure cooker for 30 min, using the rabbit anti-MT-ND2 polyclonal antibody (PA5-103952, InvitrogenTM Thermo Fisher Scientific) at a 1:200 dilution and rabbit anti-MT-CO3 antibody (HPA042788, Sigma-Aldrich ® ) at a 1:50 dilution (both incubated for 30 min at room temperature).

Techniques: Gene Expression, Derivative Assay, Mutagenesis, Comparison, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Membrane

Journal: Acta Neuropathologica

Article Title: Mitochondrial bioenergetic deficits in C9orf72 amyotrophic lateral sclerosis motor neurons cause dysfunctional axonal homeostasis

doi: 10.1007/s00401-020-02252-5

Figure Lengend Snippet: Examination of human C9orf72 post-mortem spinal cord tissue shows reduced expression of complexes I and IV of the mitochondrial electron transport chain in ventral horn motor, but not dorsal horn sensory, neurons. a Representative photomicrographs showing reduced expression of MT-ND2 (complex I) and MT-CO3 (complex IV) transcripts in ventral horn motor neurons of the post-mortem spinal cord from a C9orf72 hexanucleotide repeat expansion mutation case compared with its age- and sex-matched control. Red spots highlight individual mRNA molecules of MT-ND2 or MT-CO3. Tissue was counterstained with haematoxylin. Scale bars = 50 µm. Orange asterisk denotes an anterior horn motor neuron. Bar chart depicts the quantification of the number of MT-ND2 and MT-CO3 transcripts per ventral horn spinal motor neuron. Bars represent aggregate mean ± SEM, with each dot representing the mean count for between one and five cells for each of the five post-mortem specimens for C9orf72 -ALS (red bars) and their age- and sex-matched controls (blue bars). Statistical significance was evaluated with the Kruskal–Wallis test with FDR correction. b Representative photomicrographs of the dorsal horn sensory neurons, examined using the same probes that recognise individual mRNA of MT-ND2 or MT-CO3, showing comparable expression between the same cases and controls. Scale bars = 50 µm. Orange asterisk denotes a dorsal horn sensory neuron. Bar chart depicts the quantification of the number of MT-ND2 and MT-CO3 transcripts per dorsal horn neuron. Bars represent aggregate mean ± SEM, with each dot representing the mean count for between one and five cells for each of the five post-mortem specimens for C9orf72 -ALS (red bars) and their age- and sex-matched controls (blue bars). Statistical significance was evaluated with the Kruskal–Wallis test with FDR correction for multiple comparisons. * p < 0.05, ** p < 0.01, ‘ns’ denotes non-significant result ( p ≥ 0.05)

Article Snippet: Sections were dried overnight at 40 °C and immunostaining was performed, following epitope retrieval in citric acid buffer (pH 6) in a pressure cooker for 30 min, using the rabbit anti-MT-ND2 polyclonal antibody (PA5-103952, InvitrogenTM Thermo Fisher Scientific) at a 1:200 dilution and rabbit anti-MT-CO3 antibody (HPA042788, Sigma-Aldrich ® ) at a 1:50 dilution (both incubated for 30 min at room temperature).

Techniques: Expressing, Mutagenesis, Control